1.0 INTENDED USE
This reagent is intended for the quantitative
determination of lactate dehydrogenase (LDH) in serum.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Wroblewski and LaDue (10.2) published the first UV kinetic method for the determination of LD activity in serum in 1955. Their method was based on the classic Kubowitz and Ott (10.3) assay (1943) utilizing the pyruvate to lactate reaction. In 1956. Wacker etr al (10.4) described a procedure that followed a lactate to pyruvate reaction. The lactate to pyruvate reaction became the preferred reaction (10.5), even though the slower of the two, because of a wider linear range (10.6) and no pre-incubation requirement (10.7). The present method follows the forward reaction and has been optimized for greater sensitivity and linearity as outlined by Gay et al. (10.8)
2.2 TEST PRINCIPLE
Lactate dehydrogenase catalyzes the conversion of
L-Lactate to pyruvate in the presence of NAD.
The rate of enzymatic activity of LD is proportional to the rate of
production of NADH at 340 nm.
LDH
L‑lactate
+ NAD+ ‑‑‑---> Pyruvate + NADH + H+
Lactate dehydrogenase cat alyzes the oxidation of lactate to pyruvate with simultaneous reduction of NAD to NADH. The rate of NAD reduction can be measured as an increase in absorbance at 340nm. This rate is directly proportional to LD activity in serum.
2.3 CLINICAL SIGNIFICANCE (10.1)
Increased levels of LD are associated with myocardial
infarction. Levels reach a maximum
approximately 48 hours after the onset of pain and persist about ten days. The degree of elevation is of value in
assessing the extent of damage and in developing a prognosis. LD elevations are also observed in liver
disease, pemicious anemia, in some cases of renal disease, and in some cases of
skeletal muscle trauma. (10.1)
3.0 SPECIMEN COLLECTION AND HANDLING
3.1 PATIENT PREPARATION
No special
patient preparation is required.
3.2 SPECIMEN COLLECTION.
Fresh,
clear, unhemolyzed serum is the preferred specimen.
Serum with any visible hemolysis should not be used. Due to large amounts of LDH released from the
erythrocytes, serum should be separated from the clot as soon as possible.
Use a
standard venipuncture tube to draw patient sample.
The amount of sample required will depend on the analyzer
used. The amount of serum required is in
the range of 5-50 µl. Call Biotron's
technical service department at 1-800-595 8766 for the recommended sample volume
for your analyzer.
Record the patient's name, date and time of sample
collection and preparation.
3.3 SPECIMEN STORAGE
Serum samples can be stored at 2°-8°C for up to 3 days
with no loss of activity. (10.4) The
liver enzyme is particularly labile and is destroyed if frozen and thawed.
It is recommended that testing be done as soon as
possible after sample collection and preparation. If testing cannot occur immediately, store
the sample properly using the guidelines above.
4.0 MATERIALS (1X120,1X30 ml)
Reagents
necessary for the determination of LDH are included in the kit.
4.1 REAGENT
LDH working reagents contains:
NAD >
7.5 mM
L‑lactate
>
55 mM
sodium
azide 0.1%
buffer,
non‑ reactive stabilizers and fillers
4.2 WARNINGS AND PRECAUTIONS
For In Vitro Diagnostic Use. Not for Internal use in Humans or Animals. In Vitro Diagnostics reagents may be hazardous. Avoid ingestion and skin or eye contact. This reagent contains sodium azide (0.1%) as a preservative. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. Upon disposal, flush with large amounts of water.
4.3 REAGENT PREPARATION
The working reagent is prepared by combining 4 parts of
R1 (buffer) to 1 part of R2 (coenzyme).
4.4 REAGENT STORAGE AND STABILITY
All the reagents included in the kits are stable at 2-8°
C (refrigerated) until the expiration date stated on the labels. The working reagent is stable at 2‑8° C
(refrigerated) for 21 days.
4.5 ADDITIONAL MATERIALS REQUIRED
4.5.1 Spectrophotometer
or colorimeter capable of reading absorbance at 340 nm.
4.5.2 1 cm cuvettes
or a flow cell capable of transmitting light at 340 nm.
4.5.3 Test tube and
pipettes.
4.5.4 Timer with
one minute increments.
4.5.5 Constant
temperature source which can be adjusted to 37° C.
4.5.6 Normal and
abnormal controls for quality control.
5.0 TEST PROCEDURE
The
following is a general procedure for use on a manual instrument.
Application procedures for use on automated analyzers are
available. Contact Biotron’s Technical Service Department for specific
information.
5.1 PROCEDURE CONDITIONS
Wavelength 340
nm
Temperature 37°
C
Pathlength 1.0
cm
Mode kinetic
Lag time 30
seconds
Sample to reagent
ratio 1:20
5.2 INSTRUMENT
Any instrument capable of reading absorbance accurately
with a sensitivity of 0.001 absorbance at 340 nm may be used. The band width should be 10 nm or less, stray
light 0.5% or less, and the wavelength accuracy within 2 nm.
5.3 CALIBRATION
No reagent calibration is necessary as this procedure is
standardized based on the millimolar absorptivity of NADH which is taken as
6.22 at 340 nm under the test conditions described.
5.4 PROCEDURE
5.4.1 Prepare the
required volume of working reagent. (See
4.3 Reagent Preparation Section.)
5.4.2 Into separate
test tubes pipette 50 µl of serum to be assayed.
5.4.3 Add 1.0 ml of
working reagent, mix, and incubate for 30 seconds at 37° C.
5.4.4 Record the
increase in absorbance at 340 nm at one minute intervals until the absorbance
change is constant.
5.5 CALCULATION AND RESULTS
Lactate
dehydrogenase U/L =
DA/min X assay volume (ml) X 1000
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑------------- = DA/min X 3376
6.22 X
light path (cm) X sample volume (ml)
DA/min = change in absorbance per minute
Assay
volume = 1.050 (ml)
1000 =
converts U/ml to U/L
6.22 =
absorbance coefficient of NADH at 340 nm
Light path
= length of light path (usually 1 cm)
Sample
volume = 0.050 (ml)
3376 =
factor derived from constants in the equation
Example: Lactate dehydrogenase U/L =
0.05 X
1.050 X 1000
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑-----------
= 0.05 X 3376 = 169 U/L with DA/min = 0.05
6.22 X 1 X 0.050
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.5)
The range
of expected values is:
Male: 90‑221 U/L (37° C)
Female: 89‑187 U/L (37° C)
These values are suggested guidelines. It is recommended that each laboratory
establish the normal range for the area in which it is located.
6.2 MEDICAL ALERT VALUES (10.7)
Each laboratory should establish low and high values
beyond which the patient would require immediate attention by a physician. If a "medical alert value" is
reached, always repeat the test to confirm the result and notify a physician if
the result is confirmed.
6.3 LIMITATIONS OF PROCEDURE
Oxalates, oxamates, and EDTA inhibit LDH. Young (10.6) gives a list of drugs and other
substances that interfere with the determination of LDH activity.
7.0 QUALITY CONTROL
Standard practice for quality control should be applied
to this system. Commercially available
lyophilized controls can be used to monitor the daily acceptable
variations. Normal and abnormal controls
should be assayed at the beginning of each run of patient samples, whenever a
new reagent or a different lot number is being used, and following any system
maintenance.
A satisfactory level of performance is achieved when the
analyte values obtained are within the "acceptable range" established
by the laboratory.
8.0 CALIBRATION PROCEDURES
No routine reagent calibration is necessary as this
procedure is standardized based on the millimolar absorptivity of NADH which is
taken as 6.22 at 340 nm under the test conditions described.
9.0 PERFORMANCE CHARACTERISTICS
9.1 PRECISION
The estimates of precision shown below were obtained from assays of human control serum.
Within-Run
Mean
(U/L) SD (U/L) CV (%)
183 2.59 1.4
441 3.76 0.9
Between-Run
Mean
(U/L) SD (U/L) CV (%)
189 5.53 2.9
443 6.33 1.4
9.2 CORRELATION
A correlation study was done this method and a
comparative UV LDH method. The samples
range between 80 and 540U/L.
Number Regression Equation Correlation
of
Samples y=Biotron , x=Comparative Coefficient
103 y = 1.00 x + 7.4 0.994
9.3 LINEARITY
This procedure is linear through 1000 U/L, beyond which
the specimen should be diluted with an equal volume of deionized water. Reassay the specimen and multiply the results
by 2.
9.4 SENSITIVITY
An absorbance change of 0.001DA/min corresponds to approximately 3.4 U/L LDH activity.
10.0 REFERENCES
10.1
Tietz, N.W., editor,
Fundamentals of Clinical Chemistry, 3rd Ed., W.B. Saunders Co.,
391(1987).
10.2
Wroblewski, F., La
Due, J.S., Proc. Soc. Exp. Biol. Med. 90:210(1955).
10.3
Kubowitz, F., Ott,
P., Biochem. 314:94(1943).
10.4
Wacker, W.E.C., et
al, N. Engl. J. Med. 255:449(1956).
10.5
Henry, R.J. et al,
Clinical Chemistry; Principles and Technics, 2nd Ed., Hagerstown
(MD) Harper & Row, pp.819-831,(1974).
10.6
Amador, E., et al,
Clin. Chem. 9:391(1963).
10.7
Buhl, S.N., et al,
Clin. Chem. 23:1289(1977).
10.8
Gay, R.J., McComb,
R.B., Bowers, G.N. Clinical Chemistry, 14:740(1968).
10.9
Teitz, N.W.,
Fundamentals of Clinical Chemistry, 2nd Ed., W.B. Saunders Co.,
657,(1976).
10.10 Kreutzer, H.H., et al, Clin. Chim. Acta 9:64(1964).
10.11 NCCLS Document M29-T2, 2nd Ed.(1991).
10.12 Young, D.S., et al, Clin. Chem., 21:1D (1975).
10.13 NCCLS document “Evaluation of Precision Performance of
Clinical Chemistry Devices”, 2nd Ed.(1992).